One-third of world's population is infected with M. tuberculosis asymptomatically. Eight million new cases of active diseases develop each year & three million people succumb to this disease every year (Dye et al., 1999). With the advent of HIV & emergence of multidrug resistant strains of M. tuberculosis, the problem has increased manifold (Horsburgh, 1991; Barnes et al., 1991 and Bloch et al., 1994). The current treatment of disease usually involves combination chemotherapy based on isoniazid, pyrazinamide, rifampicin & ethambutol. In general, 6 months long course is required for effective treatment, which often results in poor compliance on the part of patients, who stop drug intake as soon as they begin to feel better. This leads to development of drug resistant forms of bacilli, which are able to survive routine drug therapy. Multidrug resistant tuberculosis (MDR-TB) is defined as a disease due to tubercle bacilli resistant to at least isoniazid and rifampicin, the two most powerful anti-tubercular drugs. Such a precarious scenario demands development of new drugs that can act on new targets and can be effective in relatively shorter periods so that the patients do not develop resistance to these drugs. The present invention can lead to the development of such target specific anti-tubercular drugs useful for short-term therapies.
Sequence analysis of various prokaryotes has shown the presence of eukaryotic like serine/threonine and tyrosine phosphatases in bacterial pathogens. In various pathogenic bacteria like Yersinia pseudotuberculosis, Salmonella typhimurium and enteropathogenic E. coli tyrosine phosphatases have been shown to act as major virulence determinants (Guan and Dixon., 1990; Galyov et al., 1993 and Kaniga et al., 1996)
YopH, one of the PTPases, is encoded by the yersiniae virulence plasmid and has been identified as an essential virulence factor (Bliska et al., 1991). YopH comprises of several domains including amino terminal sequences involved in secretion, translocation and chaperone binding; a central proline rich SH3—binding domain and a carboxyl terminal catalytic domain that is homologous to a domain in the eukaryotic PTPases (Sory et al., 1995). It is postulated that YopH disrupts a general phagocytic mechanism as both Fc receptor and complement mediated phagocytosis is inhibited by YopH. (Ruckdeschel et al., 1996 and Fallman et al., 1995). Two of the YopH substrates, p130cas and paxillin are proteins involved in connecting integrins to the actin cytoskeleton and the third one is a tyrosine kinase (Persson et al., 1997 and Black et al., 1997). The possible explanation for the role of YopH protein is that it inhibits uptake of bacteria mediated by the interaction of the bacterial outer membrane protein invasin with cellular β1 integrin. According to this model, invasin binding stimulates tyrosine phosphorylation of cellular targets, leading to cytoskeletal rearrangements and bacterial uptake. YopH dephosphorylates the protein required for this activity. Recent studies have shown that YopH also inhibits Akt pathway and phosphatidylinositol 3-kinase dependent secretion of interleukin 2 in macrophages (Sauvonnet et al., 2002).
S. typhimurium encodes a tyrosine phosphatase, SptP comprised of modular domains. The amino-terminus of SptP exhibits sequence homology to the Exotoxin S from P. aeruginosa and YopE from Yersinia spp. Exotoxin S is an ADP ribosyl transferase that has been implicated in P. aeruginosa in the induction of host cell injury and is known to be a virulence factor of P. aeruginosa. The carboxyl terminus of SptP showed homology to the eukaryotic like protein tyrosine phosphatases. The carboxyl terminus of SptP protein is homologous to YopH and the catalytic domain of the eukaryotic PTPase. The cysteine residue at position 481 is essential for its catalytic activity as mutation of this conserved cysteine residue abolishes the phosphatase activity (Kaniga et al., 1996). Kaniga et al showed that sptP mutants are defective in the colonization of spleens of orally infected BALB/c mice. SptP has been shown to possess an in vitro GTPase activating protein (GAP) activity towards two host GTP binding proteins, Rac-1 and Cdc42 that play an important role in the cytoskeletal dynamics (Fu and Galan, 1999). It has been suggested that the GAP activity of SptP could down regulate signaling through Cdc42 and Rac that could rebuild the actin cytoskeleton after Salmonella entry. Fu and Galan have shown that microinjection of purified GST-SptP into cultured cells results in the disruption of actin cytoskeleton and the disappearance of stress fibers (Fu and Galan, 1999).